Alzheimer's disease (Alzheimer's) is a common age-related brain degenerative disease. The disease is characterized by progressive dementia together with the presence of characteristic neuropathological features. The formation of .beta.-amyloid deposits or plaques is a hallmark and diagnostic feature of Alzheimer.beta.s disease (Khachaturian (1985) Arch Neurol 42:1097-1105). A significant body of evidence suggests that the process of .beta.-amyloid formation and deposition is directly linked to the development of this disease. For example, individuals with mutations in the gene encoding the .beta.-amyloid precursor protein (.beta.-APP) invariably develop Alzheimer's disease (Goate et al.(1991) Nature 353:844-846; Mullan et al. (1992) Nature Genet 1:345-347; Murrell et al. (1991) Science 254:97-99; Van Broeckhoven (1995) Eur Neurol 35:8-19). The .beta.-amyloid peptide is a 39-43 amino acid protein (Glenner et al. (1984) Biochem Biophys Res Commun 120:885-890; Masters et al (1985) Proc Natl Acad Sci USA 82:4245-4249) which is capable of forming .beta.-pleated sheet aggregates. These aggregating fibrils are subsequently deposited in the brain parenchyma or in the cerebrovasculature of the Alzheimer's disease victim.
The .beta.-amyloid peptide is derived from a larger Type I membrane spanning protein, .beta.-APP, which has several alternatively spliced transcripts (Kang et al. (1987) Nature 325:530-532; Ponte et al. (1988) Nature 331:525-527; Tanzi et al. (1988) Nature 331:528-530; Kitaguchi el al. (1988) Nature 331: 530-532; de Savage et al (1989) Science 245:651-653). The differentially spliced transcripts give rise to .beta.-APP of 695, 714, 751, and 770 amino acids. The biological function of .beta.-APP is not well understood although it appears to function in cell to cell contact, cell survival, and cell proliferation (Schubert et al. (1989) Neuron 3:689-694; Saitoh et al. (1989) Cell 58:615-622; Chen et al. (1991) Neurosci Lett 125:223-251; Mattson et al. (1993) Neuron 10:243-254).
A secreted form of .beta.-APP is normally generated by proteolytic cleavage (Weidemann et al. (1989) Cell 57:115-126). This proteolytic cleavage occurs within the .beta.-amyloid domain precluding .beta.-amyloid formation (Esch et al. (1990) Science 248:1122-1124; Sisodia et al. (1990) Science 248:492-495). As a result of the cleavage, the bulk of .beta.-APP is released from the cell and a carboxyl terminal fragment of .about.8 kDa remains bound to the cell membrane. The enzyme(s) responsible for this non-amyloidogenic processing of .beta.-APP is termed .gamma.-secretase.
The formation of .beta.-amyloid peptide is a normal physiological process. The peptide has been found to be naturally produced by cultured cells in vitro (Haass et al. (1992) Nature 359:322-325; Seubert et al (1992) Nature 359:325-327; Shoji et al. (1992) Science 258:126-129; and in vivo (Seubert et al. (1992) Nature 359:325-327; Vigo-Pelfrey et al. (1993) J Neurochem 61:1965-1968; Tabaton et al. (1994) Biochem Biophys Res Commun 200:1598-1603; Teller et al. (1996) Nature Med 2:93-95). The .beta.-amyloid peptide appears to be a degradation by-product of intracellular catabolism of the non-secreted form of .beta.-APP (Higaki et al. (1995) Neuron 14:651-659) and inhibiting its formation has no apparent deleterious consequences in vitro. There are two proteolytic processing steps required to produce the .beta.-amyloid peptide: one produces the amino-terminus of the peptide mediated by an unidentified enzyme(s) referred to as .beta.-secretase, the second forms the carboxyl-terminus of the peptide which is generated by an unidentified enyme(s) termed .gamma.-secretase.
Attention in the Alzheimer's disease research community has been directed to inhibiting the processing of .beta.-APP into .beta.-amyloid peptide as an approach to novel therapeutic development for Alzheimer's disease. Neither the .beta.- nor .gamma.-secretase processing enzymes have been definitively identified or purified. No assay exists which contains pure .beta.-APP and pure .beta.-amyloid forming enzymes. Intact cultured cells provide a source of .beta.-amyloid peptide. Screens for compounds which inhibit .beta.-amyloid production have been developed based on measurement of .beta.-amyloid production by cells in culture following application of a test compound. Toxicity of the compound is measured concomitantly. Test compounds which score as non-toxic inhibitors in such an assay are then tested for activity in animals. However, animal testing is laborious, expensive, and time consuming. In addition, many significant obstacles to obtaining inhibition of .beta.-amyloid production in an animal exist which are absent in cell culture, including metabolism and clearance of the compound, limited access to the target organ (brain) imposed by the blood brain barrier, and selective cell toxicity. Thus, negative results in animal tests are difficult to interpret and are of limited use in informing structural design of other compounds. For these reasons it would be highly desirable to obtain a system for testing compounds for .beta.-amyloid inhibiting activity and toxicity which resembled the complexity of the intact target organ, yet circumvented the technical difficulty of performing and interpreting whole animal testing experiments.
Organotypic slice culture methods have been developed for brain in which explants or sections of whole brain or, more commonly, a discrete anatomical brain structure such as hippocampus or cerebellum, are maintained in culture for extended periods of time (Seil (1979) Review in Neuroscience 4:105-177; Gahwiler (1981) J. Neurosci Meth 4:329-342; Gahwiler (1984) Neuroscience 11:751-760, Gahwiler (1988) Trends Neurosci 11:484-490). Recently significant improvements have been developed such that more reproducible results may be obtained through a simplified method (Stoppini et al., (1991) J Neurosci Methods 37:173-182). An essential feature of such cultures is the striking preservation of organotypic tissue architecture: cellular anatomy closely resembles that in the intact brain, to the extent that synaptic inputs and function mimic that of the normal situation, and development continues in neonatal brain slices. Typically, these preparations are used for electrophysiological studies investigating brain phenomena such as the biochemical basis of learning which require complex interactions of cells available only in the intact animal or organotypic slice cultures. Only two examples of use of organotypic brain slice cultures in Alzheimer's disease research are known in the art. London et al. ((1996) Proc Natl Acad Sci 93:4147-4153) looked at interactions of different brain cell types by applying monocytes pre-treated with synthetic amyloid to organotypic brain slice cultures, measuring cell survival. Nitsch, Wurtzman, and colleagues have examined the effects of neural stimulation by electrodes and neurotransmitters on brain circuitry (Diekman et al. (1994) J Neural Transm Suppl 44:61-71) and secretion of amyloid precursor protein (Nitsch et al (1994) J Neural Transm Suppl 44:21-27); Farber et al. (1995) J Neurosci 15:7442-7451).
The organotypic slice culture method has not been combined with assays for .beta.-amyloid and cell viability to examine amyloid production and/or the effect of test compounds on its production. The present invention is based, in part, on combining organotypic slice culture methods with .beta.-amyloid assays methods. The resulting methods of the present invention provides a rapid and efficient method for identifying compounds that can be used to treat Alzeimer's.
Prior art for testing compounds for inhibition of .beta.-amyloid production relied on testing activity in whole animals, primary cells or cell lines in culture, broken cell or cell or tissue homogenates, or pure enzyme preparations. With the exception of the whole animal, none of these methods mimics the complexity of cell types and interactions found in the brain. Cellular interactions are known to affect .beta.-amyloid precursor protein and .beta.-amyloid production and secretion, and can be expected to affect availability, metabolism, and tolerance of compound in brain. Organotypic brain slice culture offers the full range of cell tppes present in brain with remarkably well preserved organization and cellular interactions. Thus, effects of test compounds on .beta.-amyloid production and cellular viability and function are better predictors of in vivo effects than these measures taken on less complex single-cell systems. Candidate compounds may be eliminated prior to advancement to animal testing which have toxic effects or lack of efficacy that are evident in organotypic preparations, but which were not evident in cell culture systems.
Whole animal experiments are laborious, expensive, and time consuming to perform. In addition, relatively large amounts of test compound must be synthesized in order to dose animals. For example, under current animal testing protocols, a minimum of 7 animals/data point is generally required due to variation in animals and high sensitivity required of the assays. Each determination in organotypic culture requires a fraction of the number of animals as a similar determination in vivo: approximately 3 slices (20 are obtained/mouse or 30/rat) per data point, vs. 7 animals/data point, for a 70 fold reduction in the number of animals required. Dose response and time course studies performed in organotypic slice experiments facilitate better initial choices for in vivo dosing regimens, reducing the number of in vivo experiments with adjusted dosing regiments required.
Another major drawback of whole animal experiments in the number of variables which cannot be controlled and are difficult to assess. For example, if a compound is without effect, it may be due to rapid clearance from the blood, rapid metabolism, sequestration by a non-target tissue, or inability to penetrate the blood brain barrier. Dosing may be limited by toxicity to a sensitive non-target organ. Determining the contribution of these factors to a negative result is a major undertaking. Thus, negative results are not of use in generating structure-activity relationships to guide generation of improved compound structures. Organotypic slice culture eliminates or minimizes these variables since the blood brain barrier and other tissues are not present. Metabolism of compound is easily assessed by sampling media. Dose at the target organ is easily controlled.